Why we use functional genomics to study Pseudomonas syringae and the type III secretion system
The work in our lab focuses on the bacterium Pseudomonas syringae pv. tomato DC3000, which causes bacterial speck of tomato and has become an important model for studying general mechanisms of microbial virulence in plants. P. syringae can colonize intercellular spaces in leaves despite the fact that the plant innate immune system detects flagella and other universal features of the bacterium. The secret of P. syringae's success is the type III secretion system, which injects defense-suppressive "effector" proteins into plant cells. A given P. syringae strain (such as DC3000) is not universally virulent because in most plants one or more of the effectors will betray the pathogen to an anti-effector surveillance system that triggers a potent defense known as the hypersensitive response. A molecular arms race between microbes like P. syringae and plants has caused a remarkable proliferation in components of the attack and defense systems. DC3000 is a particularly useful pathogen for exploring the resulting multifactorial disease process because it is also capable of causing disease in the experimentally amenable model plants Arabidopsis thaliana and Nicotiana benthamiana and because its genome has been sequenced. The DC3000 genome sequence is enabling us to develop a complete "parts list" for the pathogen and to determine how these parts (for example, the ca. 30 type III effectors) work together to defeat plants.
Ultimately, this knowledge may be used to improve plant defenses against P. syringae and the many other bacterial, fungal, and oomycete pathogens that use a similar strategy involving translocated effectors to suppress innate immunity.
Our current projects are described below.
The P. syringae type III secretion system
The key to P. syringae virulence is the type III secretion system (T3SS), which has the remarkable ability to translocate effector proteins across three membranes and two walls in the journey from bacterial cytoplasm to plant cytoplasm. Many important animal pathogens also use the T3SS to inject effector proteins into host cells, but these pathogens do not have to penetrate a host cell wall. We are particularly interested in understanding how the P. syringae T3SS is adapted for translocation through plant barriers. Because of this interest we are focusing on components of the T3SS injector that are themselves extracellular and likely to interact with the plant cell wall and plasmamembrane. Genomics has helped us to identify such proteins, and we are now using a variety of genetic, biochemical, and cell biological methods to understand how they function.
The P. syringae type III effector repertoire
The type III effectors appear to mimic eukaryotic proteins as they function inside plant cells to suppress plant defenses and promote bacterial growth. No single effector is important for bacterial growth in planta, but mutants unable to deliver any effectors are nonpathogenic. We are taking a systematic approach to understanding how the complex effector repertoire functions. This involves testing all effectors in the repertoire for their ability to suppress plant defenses, to have deleterious effects when expressed in the model eukaryote yeast, to interact with key host proteins, and to affect host range, symptom production, and other disease processes. To facilitate these studies we have constructed DC3000 polymutants lacking multiple combinations of effectors.
Functional genomics of bacterial adaptations to life in planta
We are also interested in virulence factors other than the type III effectors, especially those whose expression appears to be coordinated with the type III secretion system but which have no apparent direct role in the system. Such factors include an ApbE family protein and a putative alcohol dehydrogenase, which are required for the full growth potential of DC3000 in Arabidopsis. These studies address the general question of what metabolic adaptations are needed for bacteria like P. syringae to grow in planta and whether these abilities contribute to host specificity.
We have active collaborations with several labs around the world, but three local collaborations have an almost daily impact on our program and are explained here.